Method of amplifying or detecting HIV-1 RNA

ABSTRACT

A simple, speedy and sensitive method of detecting HIV-RNA using an oligonucleotide which can bind to an intramolecularly free region of the genomic RNA of HIV-1 at relatively low and constant temperatures (at 35° C. to 50° C., preferably at 41° C.) as an oligonucleotide primer for use in amplification of a nucleic acid.

[0001] The present invention relates to oligonucleotides used foramplification or detection of HIV-1 RNA in clinical tests and diagnoses.

[0002] Human immunodeficiency virus (HIV) is the pathogen of acquiredimmunodeficiency syndrome (AIDS). Two subtypes of HIV are known: HIV-1,which is spread worldwide, and HIV-2, which is epidemic mainly on theAfrican West coast. The similarity between HIV-2 and simianimmunodeficiency virus (SIV) in base sequence implies that HIV-2 may bezoonotic. However, clinical conditions of HIV-2 infection are lessserious than those of HIV-1 infection.

[0003] HIV-1 infection induces production of antibodies againststructural proteins and regulatory proteins of HIV-1. HIV-1 attacks theT cells classified as CD4+ lymphocytes as the main target immunocytesand hence abnormalizes the immune system in various ways. In theadvanced stages of HIV-1 infection, B cells are stimulated to set offhypergammaglobulinemia, and autoantibodies and immunocomplexes appearwith marked reduction of lymphocytes and blood platelets. Complicationssuch as tuberculosis, Pneumocystis carinii pneumonia and otheropportunistic infections at high levels of immunodeficiency induced byHIV-1 infection are diagnostic of onset of AIDS.

[0004] For diagnosis of HIV-1 infection, EIA (enzyme immunoassay) basedon colorimetric detection of the reaction of an antibody against anviral antigen is available, coupled with Western blot confirmation ofsuspected positive serum samples by the presence of antibodies in theserum samples which react to a specific virus antigen in a blot ofelectrophoretically separated various virus particle antigens. However,assay methods which detect antibodies like this are not available fordiagnoses of early stage infection before production of antibodies.

[0005] As discussed above, conventional assay methods can not afforddiagnoses in the early stage of infection, require complicatedoperations and long time and can hardly detect a trace of HIV-1 in asample in a short time. Therefore, development of a speedy and sensitivedetection method is demanded. Especially, quantification of HIV-1 RNA iscrucial to get information on pathological progress and theeffectiveness of anti-HIV drugs. Further, development of automaticanalyzers is demanded to facilitate clinical tests.

[0006] For high sensitive detection, it is preferred to amplify aspecific sequence in a gene to be detected or identified or an RNAderived from such a gene before the detection. As a method of amplifyinga specific sequence in RNAs like the HIV-1 genomic RNA, the reversetranscription-polymerase chain reaction (RT-PCR) is known. In thismethod, the reverse transcription step for synthesis of the cDNA of thetarget RNA is followed by repeated cycles of heat denaturation,primer-annealing and elongation reaction in the presence of a couple ofprimers, one of which is complementary to either end of the specificsequence and the other is homologous to the other end of the specificsequence (the antisense primer may be the same as the primer used in thereverse transcription step), and a thermostable DNA polymerase to giveDNA as the amplification product of the specific sequence.

[0007] However, the necessity to conduct the operations in two steps(the reverse transcription step and the PCR step) and repeat thecumbersome operations such as rapid heating and cooling hindersautomation of the RT-PCR.

[0008] NASBA or 3SR is known as a technique for amplifying a specificRNA sequence by the cooperative action of a reverse transcriptase and anRNA polymerase. This technique activates the chain reaction comprisingsynthesis of a double-stranded DNA having a promoter sequence from thetarget RNA by using a primer having a promoter sequence, a reversetranscriptase and ribonuclease H, and formation, by an RNA polymerase,of an RNA having the specific base sequence, which is then used as thetemplate for synthesis of the above-mentioned double-stranded DNA havingthe promoter sequence, along the double-stranded DNA as the template.NASBA or 3SR allows relatively isothermal amplification of nucleic acidand is considered suitable for automation.

[0009] However, since the reactions involved in this amplificationtechnique are carried out at relatively low temperatures (for example,at 41° C.), it is possible that the formation of an intramolecularstructure of the target RNA lowers the reaction efficiency by hinderingbinding of the primers. Therefore, an operation of destroying theintramolecular structure of the target RNA such as heat denaturation ofthe target RNA is necessary before the amplification reaction toincrease the binding efficiency of the primers.

[0010] The object of the present invention is to provide a simple,speedy and sensitive method of amplifying or detecting HIV-RNA throughprovision of an oligonucleotide which can bind to an intramolecularlyfree region of the genomic RNA of HIV-1 at relatively low and constanttemperatures (at 35° C. to 50° C., preferably at 41° C.) as anoligonucleotide primer for use in amplification of a nucleic acid.

[0011] The present invention has been accomplished to attain theabove-mentioned object. The invention defined in claim 1 of the presentapplication provides a step of amplifying an RNA derived from HIV-1,which comprises synthesizing a cDNA by the action of an RNA-dependentDNA polymerase by using a specific sequence in an RNA derived from HIV-1anticipated in a sample as a template, a first primer containing asequence complementary to the specific sequence and a second primercontaining a sequence homologous to the specific sequence (either ofwhich additionally has a promoter sequence for the RNA polymerase at the5′ end), denuding the cDNA to a single-stranded DNA through degradationof the RNA in the resulting RNA-DNA double strand by ribonuclease H,forming a double-stranded DNA having a promoter sequence which can betranscribed into an RNA consisting of the specific base sequence or asequence complementary to the specific base sequence by using thesingle-stranded DNA as a template by the action of a DNA-dependent DNApolymerase, and then transcribing the double-stranded DNA into an RNAtranscript, which acts as a template in the subsequent cDNA synthesis bythe RNA-dependent DNA polymerase, in the presence of the RNA polymerase,wherein the first primer is an oligonucleotide of any one of SEQ IDNOS:1 to 7, and the second primer is an oligonucleotide of any one ofSEQ ID NOS:8 to 20.

[0012] The invention defined in claim 2 of the present applicationprovides the step according to claim 1, which further comprises adding athird oligonucleotide which is complementary to a region of the RNAderived from HIV-1 which flanks the 5′ end of the specific sequence withan overlap (of from 1 to 10 bases) with the specific sequence to form atemplate used in the initial stage of the amplification by cutting theRNA derived from HIV-1 at the 5′ end of the specific sequence (by theaction of the rebonuclease H), wherein the first primer is anoligonucleotide of any one of SEQ ID NOS:1 to 7, and

[0013] (1) the second primer is an oligonucleotide of SEQ ID NO:8, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:21 and 22,

[0014] (2) the second primer is an oligonucleotide of SEQ ID NO:9, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:22 to 26,

[0015] (3) the second primer is an oligonucleotide of SEQ ID NO:10, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:22 to 28,

[0016] (4) the second primer is an oligonucleotide of SEQ ID NO:11, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:22 to 29,

[0017] (5) the second primer is an oligonucleotide of SEQ ID NO:12, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:22 to 29,

[0018] (6) the second primer is an oligonucleotide of SEQ ID NO:13, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:23 to 30,

[0019] (7) the second primer is an oligonucleotide of SEQ ID NO:14, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:23 to 30,

[0020] (8) the second primer is an oligonucleotide of SEQ ID NO:15, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:24 to 30,

[0021] (9) the second primer is an oligonucleotide of SEQ ID NO:16, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:25 to 30,

[0022] (10) the second primer is an oligonucleotide of SEQ ID NO:17, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:27 to 31,

[0023] (11) the second primer is an oligonucleotide of SEQ ID NO:18, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:31 and 32,

[0024] (12) the second primer is an oligonucleotide of SEQ ID NO:19, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:32 and 33, or

[0025] (13) the second primer is an oligonucleotide of SEQ ID NO:20, andthe third oligonucleotide is an oligonucleotide of SEQ ID NO:33.

[0026] The invention defined in claim 3 of the present applicationprovides a step of detecting HIV-1, which comprises conducting the stepas defined in claim 1 or 2 in the presence of an oligonucleotide probe(having a sequence different from those of the first primer and thesecond primer) which can specifically bind to the RNA transcriptresulting from the amplification and is labeled with an fluorescentintercalative dye, and measuring the change in the fluorescence from thereaction solution.

[0027] The invention defined in claim 4 of the present applicationprovides the step according to claim 3, wherein the oligonucleotideprobe is designed to hybridize with at least part of the RNA transcriptand alters its fluorescence upon hybridization.

[0028] The invention defined in claim 5 of the present applicationprovides the step according to claim 4, wherein the oligonucleotideprobe has a sequence consisting of or complementary to at least 10consecutive bases in SEQ ID NO:34.

[0029]FIG. 1 is the chemical formula of the fluorescent intercalativedye moiety of the fluorescent intercalative dye-labeled oligonucleotideused in Example 2. B₁ to B₃ are nucleic acid bases.

[0030]FIG. 2 is a graph correlating the reaction time and thefluorescence enhancement accompanying RNA synthesis at initial RNAamounts of 10⁵ copies/30 μl to 10 copies/30 μl in Example 1. Negaindicates a sample prepared by using a diluent instead of the RNAsample.

[0031]FIG. 3 is a calibration curve obtained by plotting the detectiontime, which is defined as the time at the ratio of fluorescent intensityof 1.2, as ordinate and the initial RNA concentration as abscissa.

[0032]FIG. 4 is a graph correlating the reaction time and thefluorescence enhancement in the detection of HIV-RNA in the nucleic acidextracted from HIV-positive serum in Example 2.

[0033] Now, the present invention will be described in detail.

[0034] The present invention provides a nucleic acid amplification stepfor amplification of HIV-RNA in a sample, and a method of detecting theRNA transcript formed by the nucleic acid amplification step. Theamplification step of the present invention covers any amplificationmethods such as PCR, NASBA or 3SR. However, isothermal nucleic acidamplification such as NASBA or 3SR by the cooperative action of areverse transcriptase and an RNA polymerase (under such conditions thatthe reverse transcriptase and the RNA polymerase act cooperatively) ispreferred for amplifying a specific RNA sequence in HIV-1.

[0035] For example, NASBA amplification of an RNA comprises synthesizinga cDNA by the action of an RNA-dependent DNA polymerase by using aspecific sequence in HIV-1 RNA in a sample as the template, denuding thecDNA to a single-stranded DNA through degradation of the RNA in theresulting RNA-DNA double strand by ribonuclease H, forming adouble-stranded DNA having a promoter sequence which can be transcribedinto an RNA consisting of the specific base sequence or a sequencecomplementary to the specific base sequence by using the single-strandedDNA as the template by the action of a DNA-dependent DNA polymerase, andthen transcribing the double-stranded DNA into an RNA transcript, whichacts as a template in the subsequent cDNA synthesis by the RNA-dependentDNA polymerase, in the presence of an RNA polymerase. The presentinvention is characterized by the use of an oligonucleotide primer ofany one of SEQ ID NOS:1 to 7 as a first primer, which can bind to aspecific site of the HIV-1 RNA and an oligonucleotide of any one of SEQID NOS:8 to 20 as a second primer, which is homologous to part of theHIV-1 RNA to be amplified (either of which additionally has a promotersequence for the RNA polymerase at the 5′ end).

[0036] One embodiment of the present invention is the above-mentionedamplification step wherein the first primer is an oligonucleotide of anyone of SEQ ID NOS:1 to 7, and the second primer is an oligonucleotide ofany one of SEQ ID NOS:8 to 20 (provided that either the first primer orthe second primer additionally has a promoter sequence for the RNApolymerase at the 5′ end). The RNA-dependent DNA polymerase, theDNA-dependent DNA polymerase and the ribonuclease H are not particularlylimited, but AMV reverse transcriptase is preferable because it has theactivities of all of them. As the RNA polymerase, T7 phage RNApolymerase or SP6 phage RNA polymerase is preferred, though there is noparticular restriction.

[0037] In the above-mentioned amplification step, even if the specificsequence is not present at the 5′ end, HIV-1 RNA can be amplified byadding an oligonucleotide complementary to a region of HIV-1 RNA whichflanks the 5′ end of the specific sequence with an overlap (of from 1 to10 bases) with the specific sequence to cleave HIV-1 RNA at the 5′ end(by the action of a ribonuclease H) before it is used as the template inthe initial stage of the nucleic acid amplification. As the scissoroligonucleotide, an oligonucleotide of any of SEQ ID NOS:21 to 33 may beused. The scissor oligonucleotide is preferred to have a chemicallymodified hydroxyl group (for example, an aminated hydroxyl group) at the3′ end not to elongate from the 3′ end.

[0038] All When the third oligonucleotide complementary to a regionwhich flanks the 5′ end of the specific sequence with a (1 to 10-base)overlap with the specific sequence as mentioned above is added to cleaveHIV-1 RNA at the 5′ end (by the action of a ribonuclease H) before it isused as the template in the initial stage of the nucleic acidamplification, it is preferred that the first primer is anoligonucleotide of any one of SEQ ID NOS:1 to 7, and

[0039] (1) the second primer is an oligonucleotide of SEQ ID NO:8, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:21 and 22,

[0040] (2) the second primer is an oligonucleotide of SEQ ID NO:9, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:22 to 26,

[0041] (3) the second primer is an oligonucleotide of SEQ ID NO:10, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:22 to 28,

[0042] (4) the second primer is an oligonucleotide of SEQ ID NO:11, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:22 to 29,

[0043] (5) the second primer is an oligonucleotide of SEQ ID NO:12, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:22 to 29,

[0044] (6) the second primer is an oligonucleotide of SEQ ID NO:13, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:23 to 30,

[0045] (7) the second primer is an oligonucleotide of SEQ ID NO:14, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:23 to 30,

[0046] (8) the second primer is an oligonucleotide of SEQ ID NO:15, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:24 to 30,

[0047] (9) the second primer is an oligonucleotide of SEQ ID NO:16, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:25 to 30,

[0048] (10) the second primer is an oligonucleotide of SEQ ID NO:17, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:27 to 31,

[0049] (11) the second primer is an oligonucleotide of SEQ ID NO:18, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:31 and 32,

[0050] (12) the second primer is an oligonucleotide of SEQ ID NO:19, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:32 and 33, or

[0051] (13) the second primer is an oligonucleotide of SEQ ID NO:20, andthe third oligonucleotide is an oligonucleotide of SEQ ID NO:33.

[0052] In this case, the third oligonucleotide (the scissoroligonucleotide) is preferred to have a chemically modified hydroxylgroup (for example, an aminated hydroxyl group) at the 3′ end not toelongate from the 3′ end, too.

[0053] Detection of the amplification product obtained in the nucleicacid amplification step is preferably carried out by measuring thechange in the fluorescence of the reaction solution during theamplification step in the presence of an oligonucleotide probe labeledwith a fluorescent intercalative dye, though it can be detected byconventional methods for detection of nucleic acid. The oligonucleotideprobe may be, for example, an oligonucleotide having a fluorescentintercalative dye linked to a phosphorus atom via a linker. Such apreferable probe alters its fluorescence upon formation of a doublestrand with the target nucleic acid (a complementary nucleic acid)through intercalation of the intercalator moiety to the double strand(Ishiguro, T. et al., (1996) Nucleic Acids Res. 24 (24) 4992-4997).

[0054] The sequence of the probe is not particularly limited as long asit contains a sequence complementary to at least part of the RNAtranscript. For example, when the combination of a first primer of anyone of SEQ ID NOS:1 to 7 and a second primer of any one of SEQ ID NOS:8to 20, is used in the RNA amplification step, a sequence consisting ofor complementary to at least 10 consecutive bases in SEQ ID NO:34 ispreferred. In this case, it is also preferred to chemically modify thehydroxyl group at the 3′ end of the probe (for example, by addingglycolic acid) to prevent elongation reaction using the probe as aprimer.

[0055] By carrying out the amplification step in the presence of theabove-mentioned probe, amplification and detection of HIV-1 RNA can becarried out at constant temperature in one tube in one step and can beautomated easily.

[0056] Now, the present invention will be described in further detail byreferring to Examples. However, the present invention is by no meansrestricted to these specific Examples.

EXAMPLE 1

[0057] The target HIV-RNA derived from various numbers of initial copieswas detected using the combinations of oligonucleotides in the presentinvention.

[0058] (1) A 1628-nt RNA base sequence in the HIV-1 RNA base sequencecontaining the structural gene of the core protein (gag) was used as astandard RNA. The standard RNA was obtained from HIV-1 RNA with ACCRUN™315 (product name), HIV-1 RNA Positive Control, Series 400 (BBI (BostonBiomedica, Inc.) by conventional extraction and synthesis of adouble-stranded DNA containing the base sequence of the gag region byRT-PCR and in vitro transcription using the DNA as the template andpurified.

[0059] (2) The standard RNA was quantified by UV absorptiometry at 260nm and diluted with an RNA diluent (10 mM Tris-HCl (pH 8.0), 0.1 mMEDTA, 1 mM DTT, 0.5 U/μl RNase inhibitor (Takara Shuzo Co., Ltd.)) to10⁵ copies/5 μl, 10⁴ copies/ 5 μl, 10³ copies/5 μl, 10² copies/5 μl and10 copies/5 μl. The diluent alone was used as a control sample (Nega).

[0060] (3) 20.8 μl portions of a reaction solution of the followingcomposition were dispensed into 0.5 ml PCR tubes (Gene Amp Thin-WalledReaction Tubes, Perkin Elmer), and 5 μl of the RNA sample at theabove-mentioned concentrations was added.

[0061] The composition of the reaction solution (in terms of theconcentrations in the final volume of 30 μl)   60 mM Tris-HC1 buffer (pH8.6)   13 mM Magnesium chloride  115 mM Potassium chloride   39 U RNaseInhibitor   1 mM DTT 0.25 mM each of dATP, dCTP, dGTP and dTTP  3.6 mMITP  3.0 mM each of ATP, CTP, GTP and UTP  1.0 μM First primer (SEQ IDNO: 1)  1.0 μM Second primer (SEQ ID NO: 15) (which had a base sequence(SEQ ID NO: 35) including the T7 promoter sequence at the 5′ end (SEQ IDNO: 35 comprises the T7 promoter sequence from “A” at position 1 fromthe 5′ end to “A” at position 22 and the subsequent enhancer sequencefrom “G” at position 23 to “A” at position 28) 0.16 μM Thirdoligonucleotide (SEQ ID NO: 27)   25 nN Oligonucleotide (SEQ ID NO: 34)labeled with a fluorescent intercalative dye (FIG. 1) (having thefluorescent intercalative dye between “T” at position 14 from the 5′ endand “T” at position 15 and having a hydroxyl group modified withglycolic acid at the 3′ end)   13% DMSO Distilled water for volumeadjustment

[0062] (4) The reaction solutions were incubated at 41° C. for 5minutes, and 4.2 μl of an enzyme solution of the following compositionwhich was pre-incubated at 41° C. for 2 minutes was added.

[0063] The composition of the enzyme solution (in terms of theconcentrations in the final volume of 30 μl)  1.7% Sorbitol  3 μg Bovineserum albumin 142 U T7 RNA polymerase (GIBCO)  8 U AMV reversetranscriptase (Takara Shuzo Co., Ltd.) Distilled water for volumeadjustment

[0064] (5) The fluorescence intensities of the reaction solutions in thePCR tubes were directly monitored at 41° C. in a thermostaticfluorescent spectrophotometer at an excitation wavelength of 470 nm andan emission wavelength of 510 nm. The time courses of the ratio offluorescence intensities of the samples (fluorescence intensity at acertain time/background fluorescence intensity) from addition of theenzyme solution at 0 minute were shown in FIG. 2. The initial amounts ofthe RNA were from 10 copies/30 μl to 10⁵ copies/30 μl.

[0065] As shown in FIG. 2, the fluorescence profile was dependent on theinitial concentration of the standard RNA, and it was possible to detect10 copies in about 10 minutes. When the detection time, which is definedas the time at the ratio of fluorescent intensity of 1.2, was plotted asordinate, and the initial RNA concentration was plotted as abscissa, alinear relation was found between them (FIG. 3), and it was indicatedthat it is possible to quantify HIV-1 RNA in an unknown sample by usingFIG. 3 as the calibration curve. Thus, it is proved that the presentinvention allows speedy and sensitive quantitative detection of HIV-1RNA.

EXAMPLE 2

[0066] HIV-1 RNA in the nucleic acid extracted from HIV-positive serumwas detected using the combinations of oligonucleotides in the presentinvention.

[0067] (1) As the HIV-positive serum, ACCRUN™ 315 (product name), HIV-1RNA Positive Control, Series 400 (BBI (Boston Biomedica, Inc.) was used.HIV-1 RNA was obtained from 30 μl of the positive serum by conventionalextraction and diluted with an RNA diluent (10 mM Tris-HCl (pH 8.0), 0.1mM EDTA, 1 mM DTT, 0.5 U/μl RNase inhibitor(Takara Shuzo Co., Ltd.)) toan estimated RNA amount of 10³ copies/5 μl. The same standard RNA as inExample 1 was used at a concentration of 10³ copies/5 μl.

[0068] (2) A 20.8 μl portion of a reaction solution of the followingcomposition was dispensed into a 0.5 ml PCR tube (Gene Amp Thin-WalledReaction Tubes, Perkin Elmer), and 5 μl of the RNA sample at theabove-mentioned concentration was added.

[0069] The composition of the reaction solution (in terms of theconcentrations in the final volume of 30 μl)   60 mM Tris-HC1 buffer (pH8.6)   13 mM Magnesium chloride  115 mM Potassium chloride   39 U RNaseInhibitor   1 mM DTT 0.25 mM each of dATP, dCTP, dGTP and dTTP  3.6 mMITP  3.0 mM each of ATP, CTP, GTP and UTP  1.0 μM First primer (SEQ IDNO: 2)  1.0 μM Second primer (SEQ ID NO: 13) (which had a base sequence(SEQ ID NO: 35) including the T7 promoter sequence at the 5′ end (SEQ IDNO: 35 comprises the T7 promoter sequence from “A” at position 1 fromthe 5′ end to “A” at position 22 and the subsequent enhancer sequencefrom “G” at position 23 to “A” at position 28) 0.16 μM Thirdoligonucleotide (SEQ ID NO: 26)   25 nM Oligonucleotide (SEQ ID NO: 34)labeled with a fluorescent intercalative dye (FIG. 1) (having thefluorescent intercalative dye between “T” at position 14 from the 5′ endand “T” at position 15 and having a hydroxyl group modified withglycolic acid at the 3′ end)    13% DMSO Distilled water for volumeadjustment

[0070] (3) The reaction solution was incubated at 41° C. for 5 minutes,and 4.2 μl of an enzyme solution of the following composition which waspre-incubated at 41° C. for 2 minutes was added.

[0071] The composition of the enzyme solution (in terms of theconcentrations in the final volume of 30 μl)  1.7% Sorbitol  3 μg Bovineserum albumin 142 U T7 RNA polymerase (GIBCO)  8 U AMV reversetranscriptase (Takara Shuzo Co., Ltd.) Distilled water for volumeadjustment

[0072] (4) The fluorescence instensity of the reaction solution in thePCR tube was directly monitored at 41° C. in a thermostatic fluorescentspectrophotometer at an excitation wavelength of 470 nm and an emissionwavelength of 510 nm. The time course of the ratio of fluorescenceintensity of the sample (fluorescence intensity at a certaintime/background fluorescence intensity) from addition of the enzymesolution at 0 minute was shown in FIG. 4.

[0073]FIG. 4 demonstrates that it was possible to detect the RNA (at anestimated concentration of 10³ copies/30 μl) extracted from theHIV-positive serum at the same detection time as the standard RNA (RNAconcentration: 10³ copies/30 μl) as the control.

[0074] Thus, it is proved that the present invention allows speedy andsensitive detection of HIV-1 RNA extracted from HIV-positive serum.

[0075] As described above, the present invention provide a simple,speedy and sensitive method of detecting HIV-RNA through provision of anoligonucleotide which can bind to an intramolecularly free region of thegenomic RNA of HIV-1 at relatively low and constant temperatures (at 35°C. to 50° C., preferably at 41° C.) as an oligonucleotide primer for usein amplification of a nucleic acid.

[0076] The entire disclosure of Japanese Patent Application No.2001-129210 filed on Apr. 26, 2001 including specification, claims,drawings and summary are incorporated herein by reference in itsentirety.

1 35 1 20 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 1 actgtattat ataatgatct20 2 20 DNA Artificial Sequence SYNTHETIC DNA 2 attatataat gatctaagtt 203 20 DNA Artificial Sequence SYNTHETIC DNA 3 ataatgatct aagttcttct 20 420 DNA Artificial Sequence SYNTHETIC DNA 4 gagggttgct actgtattat 20 5 20DNA Artificial Sequence SYNTHETIC DNA 5 caatagaggg ttgctactgt 20 6 20DNA Artificial Sequence SYNTHETIC DNA 6 gcacacaata gagggttgct 20 7 20DNA Artificial Sequence SYNTHETIC DNA 7 ttgctactgt attatataat 20 8 25DNA Artificial Sequence SYNTHETIC DNA 8 ggaaagaaaa aatataaatt aaaac 25 925 DNA Artificial Sequence SYNTHETIC DNA 9 gaaaaaatat aaattaaaac atata25 10 25 DNA Artificial Sequence SYNTHETIC DNA 10 aaaaatataa attaaaacatatagt 25 11 25 DNA Artificial Sequence SYNTHETIC DNA 11 aaaatataaattaaaacata tagta 25 12 25 DNA Artificial Sequence SYNTHETIC DNA 12aaatataaat taaaacatat agtat 25 13 25 DNA Artificial Sequence SYNTHETICDNA 13 aatataaatt aaaacatata gtatg 25 14 25 DNA Artificial SequenceSYNTHETIC DNA 14 atataaatta aaacatatag tatgg 25 15 25 DNA ArtificialSequence SYNTHETIC DNA 15 tataaattaa aacatatagt atggg 25 16 25 DNAArtificial Sequence SYNTHETIC DNA 16 ataaattaaa acatatagta tgggc 25 1725 DNA Artificial Sequence SYNTHETIC DNA 17 aaattaaaac atatagtatg ggcaa25 18 25 DNA Artificial Sequence SYNTHETIC DNA 18 aaaacatata gtatgggcaagcagg 25 19 25 DNA Artificial Sequence SYNTHETIC DNA 19 atatagtatgggcaagcagg gagct 25 20 25 DNA Artificial Sequence SYNTHETIC DNA 20gtatgggcaa gcagggagct agaac 25 21 20 DNA Artificial Sequence SYNTHETICDNA 21 tttccccctg gccttaaccg 20 22 20 DNA Artificial Sequence SYNTHETICDNA 22 ttttctttcc ccctggcctt 20 23 20 DNA Artificial Sequence SYNTHETICDNA 23 ttttttcttt ccccctggcc 20 24 20 DNA Artificial Sequence SYNTHETICDNA 24 attttttctt tccccctggc 20 25 20 DNA Artificial Sequence SYNTHETICDNA 25 tattttttct ttccccctgg 20 26 20 DNA Artificial Sequence SYNTHETICDNA 26 atattttttc tttccccctg 20 27 20 DNA Artificial Sequence SYNTHETICDNA 27 tatatttttt ctttccccct 20 28 20 DNA Artificial Sequence SYNTHETICDNA 28 ttatattttt tctttccccc 20 29 20 DNA Artificial Sequence SYNTHETICDNA 29 tttatatttt ttctttcccc 20 30 20 DNA Artificial Sequence SYNTHETICDNA 30 aatttatatt ttttctttcc 20 31 20 DNA Artificial Sequence SYNTHETICDNA 31 gttttaattt atattttttc 20 32 20 DNA Artificial Sequence SYNTHETICDNA 32 tatatgtttt aatttatatt 20 33 20 DNA Artificial Sequence SYNTHETICDNA 33 catactatat gttttaattt 20 34 19 DNA Artificial Sequence SYNTHETICDNA 34 tctgaaggga tggttgtag 19 35 28 DNA Artificial Sequence SYNTHETICDNA 35 aattctaata cgactcacta tagggaga 28

1. A step of amplifying an RNA derived from HIV-1, which comprisessynthesizing a cDNA by the action of an RNA-dependent DNA polymerase byusing a specific sequence in an RNA derived from HIV-1 anticipated in asample as a template, a first primer containing a sequence complementaryto the specific sequence and a second primer containing a sequencehomologous to the specific sequence (either of which additionally has apromoter sequence for the RNA polymerase at the 5′ end), denuding thecDNA to a single-stranded DNA through degradation of the RNA in theresulting RNA-DNA double strand by ribonuclease H, forming adouble-stranded DNA having a promoter sequence which can be transcribedinto an RNA consisting of the specific base sequence or a sequencecomplementary to the specific base sequence by using the single-strandedDNA as a template by the action of a DNA-dependent DNA polymerase, andthen transcribing the double-stranded DNA into an RNA transcript, whichacts as a template in the subsequent cDNA synthesis by the RNA-dependentDNA polymerase, in the presence of the RNA polymerase, wherein the firstprimer is an oligonucleotide of any one of SEQ ID NOS:1 to 7, and thesecond primer is an oligonucleotide of any one of SEQ ID NOS:8 to
 20. 2.The step according to claim 1, which further comprises adding a thirdoligonucleotide which is complementary to a region of the RNA derivedfrom HIV-1 which flanks the 5′ end of the specific sequence with anoverlap (of from 1 to 10 bases) with the specific sequence to form atemplate used in the initial stage of the amplification by cutting theRNA derived from HIV-1 at the 5′ end of the specific sequence (by theaction of the rebonuclease H), wherein the first primer is anoligonucleotide of any one of SEQ ID NOS:1 to 7, and (1) the secondprimer is an oligonucleotide of SEQ ID NO:8, and the thirdoligonucleotide is an oligonucleotide of any one of SEQ ID NOS:21 and22, (2) the second primer is an oligonucleotide of SEQ ID NO:9, and thethird oligonucleotide is an oligonucleotide of any one of SEQ ID NOS:22to 26, (3) the second primer is an oligonucleotide of SEQ ID NO:10, andthe third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:22 to 28, (4) the second primer is an oligonucleotide of SEQ IDNO:11, and the third oligonucleotide is an oligonucleotide of any one ofSEQ ID NOS:22 to 29, (5) the second primer is an oligonucleotide of SEQID NO:12, and the third oligonucleotide is an oligonucleotide of any oneof SEQ ID NOS:22 to 29, (6) the second primer is an oligonucleotide ofSEQ ID NO:13, and the third oligonucleotide is an oligonucleotide of anyone of SEQ ID NOS:23 to 30, (7) the second primer is an oligonucleotideof SEQ ID NO:14, and the third oligonucleotide is an oligonucleotide ofany one of SEQ ID NOS:23 to 30, (8) the second primer is anoligonucleotide of SEQ ID NO:15, and the third oligonucleotide is anoligonucleotide of any one of SEQ ID NOS:24 to 30, (9) the second primeris an oligonucleotide of SEQ ID NO:16, and the third oligonucleotide isan oligonucleotide of any one of SEQ ID NOS:25 to 30, (10) the secondprimer is an oligonucleotide of SEQ ID NO:17, and the thirdoligonucleotide is an oligonucleotide of any one of SEQ ID NOS:27 to 31,(11) the second primer is an oligonucleotide of SEQ ID NO:18, and thethird oligonucleotide is an oligonucleotide of any one of SEQ ID NOS:31and 32, (12) the second primer is an oligonucleotide of SEQ ID NO:19,and the third oligonucleotide is an oligonucleotide of any one of SEQ IDNOS:32 and 33, or (13) the second primer is an oligonucleotide of SEQ IDNO:20, and the third oligonucleotide is an oligonucleotide of SEQ IDNO:33.
 3. A step of detecting HIV-1, which comprises conducting the stepas defined in claim 1 or 2 in the presence of an oligonucleotide probe(having a sequence different from those of the first primer and thesecond primer) which can specifically bind to the RNA transcriptresulting from the amplification and is labeled with an fluorescentintercalative dye, and measuring the change in the fluorescence from thereaction solution.
 4. The step according to claim 3, wherein theoligonucleotide probe is designed to hybridize with at least part of theRNA transcript and alters its fluorescence upon hybridization.
 5. Thestep according to claim 4, wherein the oligonucleotide probe has asequence consisting of or complementary to at least 10 consecutive basesin SEQ ID NO:34.